Spironolactone (SL) is widely used as a aldosterone antagonist in the treatment of congestive heart failure, cirrhosis, and essential hypertension. Among the side effects of SL are interference with steroid hormone production and/or metabolism and hepatic enzyme induction. The whole body metabolism of SL in vivo has been extensively studied but accurate indentification and quantitation of metabolites has been limited by the techniques employed. In addition, little is known about the sites of metabolism or disposition of SL in target tissues. The latter is of particular interest since metabolites appear to be important in both the therapeutic actions and side effects of SL. The overall objective of the proposed investigations is to characterize the pathways and products of SL metabolism at a number of sites relevant to its actions. SL metabolism will be studied in adrenals, testes, kidneys, liver, and blood in guinea pigs and rats using high pressure liquid chromatography (HPLC) and mass spectrometry. SL will be incubated in vitro with isolated whole cells or subcellular fractions (microsomes, mitochondria, cytosol) obtained from the various tissues. The identity of the metabolites produced in vitro as well as the kinetics of metabolism and cofactor requirements will be determined. The role of mixed function oxidases (MFO) in SL metabolism will be studied using MFO inhibitors in vitro or pretreatment with inducing agents (including SL) in vivo. In additiona, radioactively labelled SL will be administered to animals and the tissue distribution, subcellular distribution within tissues, identity, and covalent binding of tissue metabolites determined at various times thereafter. The effects of pretreatment with MFO inducing agents or inhibitors, or with SL itself, on the distribution and metabolism of SL will similarly be evaluated. The studies proposed will help to establish the role of metabolites in the therapeutic actions as well as the side effects of SL and may contribute to the development of more effective drugs with fewer untoward effects.